6,6',6''-(1,3,5-triazine-2,4,6-triyltriimino)tris-hexanoic acid, compd. with 1-amino-2-propanol

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 September 2017 to 17 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

DEFINITIVE TEST
- Samples were taken from the bulk test preparation of the control and each test group at 0 hours.
- Samples were also taken from the pooled replicates at 48 hours for quantitative analysis.
- Samples were stored frozen prior to analysis.
- Duplicate samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.
- Only samples at the No Observed Effect Concentration were analysed.

Vehicle:

no

Details on test solutions:

TEST WATER
- Reconstituted water (ISO medium) used for the range-finding is defined in Annex 4 (attached).
- The definitive test was performed in Elendt M7 (see Annex 2, attached).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST SYSTEM
- The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
- Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see Annex 2, attached) in a temperature controlled room maintaining the water temperature at 18 to 22 °C.
- The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin flake food suspension.
- Culture conditions ensured that reproduction was by parthenogenesis.
- Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

Reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3 (see Annex 4, attached).

Test temperature:

19 °C to 21 °C

pH:

7.8 to 8.0 (see Table 3, attached)

Dissolved oxygen:

8.4 to 9.3 mg O2/L (see Table 3, attached)

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal concentrations of 10, 18, 32, 56 and 100 mg a.i./L

Details on test conditions:

RANGE FINDING TEST
- The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.
- A nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 L to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 0.10, 1.0 and 10 mg/L.
- Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test 5 daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised daphnids were recorded.
- The control group was maintained under identical conditions but not exposed to the test item.
- A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.

DEFINITIVE TEST
- After the conduct of the range-finding test, information pertaining to the purity of the test item was obtained. At the request of the Sponsor, for the purposes of the definitive test, all test concentrations were corrected for a test item water content of 41%.
- Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg a.i./L.
- A nominal amount of test item (340 mg) was dissolved in test water and the volume adjusted to 2 L to give the 100 mg a.i./L test concentration from which a series of dilutions was made to give further test concentrations of 10, 18, 32 and 56 mg a.i./L.
- Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours (see Annex 5, attached).

EXPOSURE CONDITIONS
- As in the range-finding test 150 mL glass beakers containing approximately 100 mL of test preparation were used.
- At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations.
- Four replicate test and control vessels were prepared.
- The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ± 1 °C with a photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
- The control group was maintained under identical conditions but not exposed to the test item.
- The test preparations were not renewed during the exposure period.

TEST ORGANISM OBSERVATIONS
- Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure.
- The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

WATER QUALITY CRITERIA
- Water temperature was recorded daily throughout the test using a Hanna Instruments H93510 digital thermometer.
- Dissolved oxygen concentrations and pH were recorded at the start and termination of the test using a Hach Flexi handheld meter.
- Light intensity during the light period was measured using an ATP Instruments Lux meter.
- The appearance of the test media was recorded daily.

VALIDATION CRITERIA
- Results of the test are considered valid if the following performance criteria are met:
(a) No more than 10 % of the control daphnids show immobilisation or other signs of disease or stress (e.g. discoloration or unusual behaviour such as trapping at the surface water).
(b) The dissolved oxygen concentration at the end of the test is ≥ 3 mg/L in the control and test vessels.

MAJOR COMPUTERISED SYSTEMS
- Building management: Delta Control System

Reference substance (positive control):

yes

Remarks:

potassium dichromate (test performed between 20 October 2016 and 22 October 2016; see Annex 3, attached)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

RANGE FINDING TEST
- Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 (attached).
- No immobilisation was observed at the test concentrations of 0.10, 1.0, 10 and 100 mg/L, however, sub-lethal effects were observed at 1.0, 10 and 100 mg/L.
- Based on this information test concentrations of 10, 18, 32, 56, and 100 mg a.i./L were selected for the definitive test.
- Chemical analysis of the 100 mg/L test preparation at 0 hours (see Annex 5, attached) showed a measured test concentration of 105 % of nominal. There was no significant change in the measured concentration at 48 hours indicating that the test item was stable under test conditions.

DEFINITIVE TEST VERIFICATION OF TEST CONCENTRATIONS
- Analysis of the test preparations at 0 and 48 hours (see Annex 5) showed measured test concentrations to range from 96 % to 98 % of nominal concentrations and so the results are based on nominal test concentrations.

IMMOBILISATION DATA
- Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2 (attached).
- There was no immobilization in 20 daphnids exposed to a test concentration of 100 mg a.i./L for a period of 48 hours.
- Exposure of Daphnia magna to the test item gave EC50 values of greater than 100 mg a.i./L. The No Observed Effect Concentration was 100 mg a.i./L.

SUB-LETHAL EFFECTS
- No sub-lethal effects of exposure were observed throughout the test.

VALIDATION CRITERIA
- The test was considered to be valid given that none of the control daphnids showed immobilisation or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

WATER QUALITY CRITERIA
- The results of the water quality measurements are given in Table 3 (attached).
- Temperature was maintained at 19 °C to 21 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
- Throughout the test the light intensity was observed to be in the range 372 to 482 Lux.

OBSERVATIONS ON TEST ITEM SOLUBILITY
- At the start and throughout the test all control and test solutions were observed to be clear colourless solutions.

Results with reference substance (positive control):

- Results from the positive control with potassium dichromate were within the normal range for the reference item.

Reported statistics and error estimates:

STATISTICAL ANALYSIS
- An estimate of the EC50 values was given by inspection of immobilisation data.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item gave EC50 values of greater than 100 mg a.i./L. The No Observed Effect Concentration was 100 mg a.i./L.

Executive summary:

GUIDELINE

A study was performed to assess the acute toxicity of the test item toDaphnia magna.The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

 

METHODS

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100 mg a.i./L for 48 hours at a temperature of 19 °C to 21 °C under static test conditions. The number of immobilised daphnia were recorded after 24 and 48 hours.

 

RESULTS

Analysis of the test preparations at 0 and 48-Hours showed measured test concentrations to range from 96 % to 98 % of nominal concentration and so the results were based on nominal test concentrations.

 

CONCLUSION

Exposure of Daphnia magna to the test item gave EC50 values of greater than 100 mg a.i./L. The No Observed Effect Concentration was 100 mg a.i./L.

Description of key information

Exposure of Daphnia magna to the test item gave EC50 values of greater than 100 mg a.i./L. The No Observed Effect Concentration was 100 mg a.i./L (OECD 202 and EU Method C.2).

Additional information

GUIDELINE

A study was performed to assess the acute toxicity of the test item toDaphnia magna.The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

 

METHODS

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100 mg a.i./L for 48 hours at a temperature of 19 °C to 21 °C under static test conditions. The number of immobilised daphnia were recorded after 24 and 48 hours.

 

RESULTS

Analysis of the test preparations at 0 and 48-Hours showed measured test concentrations to range from 96 % to 98 % of nominal concentration and so the results were based on nominal test concentrations.

 

CONCLUSION

Exposure of Daphnia magna to the test item gave EC50 values of greater than 100 mg a.i./L. The No Observed Effect Concentration was 100 mg a.i./L.